Decreased Jumonji Domain-Containing 3 at the Promoter Downregulates Hematopoietic Progenitor Kinase 1 Expression and Cytoactivity of T Follicular Helper Cells from Systemic Lupus Erythematosus Patients

T follicular helper (Tfh) cells are overactivated in systemic lupus erythematosus (SLE) patients and contribute to excessive immunity. Hematopoietic progenitor kinase 1 (HPK1), as an inhibitor of T cells, is underexpressed in SLE Tfh cells and consequently induces autoimmunity. However, the reason for downregulation of HPK1 in SLE Tfh cells remains elusive. By combining chromatin immunoprecipitation with quantitative polymerase chain reaction assays, it was found that histone H3 lysine 27 trimethylation (H3K27me3) at the HPK1 promoter in SLE Tfh cells elevated greatly. We also confirmed jumonji domain-containing 3 (JMJD3) binding at the HPK1 promoter in SLE Tfh cells reduced profoundly. Knocking down JMJD3 in normal Tfh cells with siRNA alleviated enrichments of JMJD3, H3K4me3, and mixed-lineage leukemia (MLL) 1 at the HPK1 promoter and increased H3K27me3 number in the region. HPK1 expression was lowered, while Tfh cell proliferation activity, IL-21 and IFNγ secretions in the supernatants of Tfh cells, and IgG1 and IgG3 concentrations in the supernatants of Tfh-B cell cocultures all upregulated markedly. In contrast, elevating JMJD3 amount in SLE Tfh cells by JMJD3-overexpressed plasmid showed opposite effects. The abundances of H3K4me3 and MLL1 at the HPK1 promoter in SLE Tfh cells were greatly attenuated. Our results suggest that deficient JMJD3 binding at the promoter dampens HPK1 expression in SLE Tfh cells, thus making Tfh cells overactive, and ultimately results in onset of SLE.


Introduction
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, which may seriously threaten public health [1]. The basic pathogenesis of SLE is related to overactivation of T cells, which leads to overstimulation of B cells, release of inflammatory factors and autoantibodies, and autoimmune responses to multiple organs and systems [2]. However, its specific pathogenesis still remains elusive.
In recent years, a new subgroup of T cells, T follicular helper (Tfh) cells, has been discovered. They are specialized providers of T cells assisting B cells to differentiate, mature, produce high-affinity antibodies, and form germinal center (GC) [3][4][5][6]. Studies have shown that Tfh cells play a crucial role in the pathogenesis of SLE. It has been confirmed that Tfh cells account for a significantly higher proportion in peripheral blood circulation of SLE patients, and they are positively correlated with SLE disease activity index (SLE-DAI), antinuclear antibody (ANA), and anti-double stranded DNA (dsDNA) antibody levels in serum and are negatively correlated with complement C3 concentration [7,8]. The secretions of interleukin-21 (IL-21), B cellactivating factor (BAFF), interferon-γ (IFNγ), and IL-17A from Tfh cells can promote the development of SLE [9][10][11][12]. Therefore, Tfh cells have become a research hotspot in the pathogenesis of SLE.
Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine protein kinase [13,14]. It is involved in various immune responses, such as promoting the activation of c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB), degrading inhibitor of NF-κB (IκB), regulating cell proliferation and apoptosis, and inhibiting T cell-mediated immune responses and T cell receptor (TCR) signaling [14][15][16]. Our previous study revealed that the expression of HPK1 in Tfh cells dramatically decreased in SLE patients, leading to overactivation of Tfh cells, and therefore, more IL-21, BAFF, and IFNγ were produced and B cells were stimulated to produce more immunoglobulin G (IgG) [17]. However, the reason for reduced expression of HPK1 in SLE Tfh cells has not been clarified.
Our previous study revealed that H3K27me3 level at the HPK1 promoter in CD4 + T cells of SLE patients significantly decreased [2]. However, the above-mentioned study only involved HPK1 at the CD4 + T cellular level. We guessed whether the enrichments of EZH2, JMJD3, or UTX within the HPK1 promoter region of SLE Tfh cells altered and therefore contributed to a series of epigenetic changes. All these alleviated HPK1 expression and stimulated Tfh cell overactivation. At last, overactivated Tfh cells promoted autoimmune response of SLE. This research will further uncover the epigenetic pathogenesis of SLE and is expected to provide new insights into the treatment of SLE.

Methods and Materials
2.1. Subjects. A total of 30 patients with SLE (SLE group; age, 28:93 ± 5:75 years old) were enrolled from the out-patient clinic and in-patient ward of the Department of Dermatology, the Second Xiangya Hospital, Central South University (Changsha, China). The data of patients are listed in Table 1. Besides, 30 healthy controls (control group; age, 27:80 ± 5:51 years) were recruited from healthy staff and graduate students of the Second Xiangya Hospital, and their data are pre-sented in Table 2. The SLE group and control group were age and sex matched, and written informed consent was obtained from each participant before enrollment. The study was performed according to the Declaration of Helsinki, and it was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University.

Cell
Isolation. Every participant's venous peripheral blood was preserved in heparin. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by Ficoll-Hypaque density gradient centrifugation (GE Healthcare). Whereafter, naive CD4 + T cells were isolated by human naive CD4 + T cell isolation kit (Miltenyi), and B cells were collected using CD19 magnetic beads (Miltenyi), according to the protocols of the manufacturer.
2.5. RNA Isolation and cDNA Synthesis. Total RNA of Tfh cells was isolated by a RNeasy mini kit (Qiagen), according to the manufacturer's instruction. Subsequently, cDNA was synthesized from 1 μg of total RNA with a miScript Reverse Transcription kit (Qiagen) and stored at −80°C.
2.6. QPCR. The qPCR was carried out with a Rotor-Gene 3000 thermocycler (Corbett Research Ltd.). The expression of target cDNA was measured by a SYBR Premix Ex Taq kit (TaKaRa), and β-actin was used as an endogenous control. A series of five dilutions from a cDNA sample were simultaneously amplified to generate a standard curve, in order to evaluate the relative amount of each cDNA sample. The primers used in this study were as follows: for HPK1, 5 ′ -CTGCTGGAACGGAAAGAGAC-3 ′ (forward) and 5 ′ -CGGACAAGCAGGAATTTGTT-3 ′ (reverse); for 2 Journal of Immunology Research β-actin, 5 ′ -CGCGAGAAGATGACCCAGAT-3 ′ (forward) and 5 ′ -GCACTGTGTTGGCGTACAGG-3 ′ (reverse).

Western Blot
Analysis. Tfh cells were lysed by whole-cell lysis buffer (Thermo Fisher Scientific). The lysates were centrifuged, and the proteins were quantified using a Bradford protein assay kit (Bio-Rad Laboratories Inc.). Subsequently, the proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with 8% polyacrylamide gels and were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with Tris-buffered saline-Tween (TBST) buffer supplemented with 5% nonfat dry milk and incubated at 4°C overnight with HPK1 antibody (dilution, 1 : 100; Santa Cruz Biotechnology Inc.), JMJD3 antibody (dilution, 1 : 100; Abgent Ltd.), or β-actin antibody (dilution, 1 : 100; Santa Cruz Biotechnology Inc.). The blots were exposed to X-ray films, and the band densities were analyzed by the Quantity One Analysis software (Bio-Rad Laboratories Inc.).

Transfection.
Control-siRNA and JMJD3-siRNA were designed and synthesized by Guangzhou RiboBio in China. The pcDNA3.1 blank plasmid and pcDNA3.1-JMJD3expressing plasmid were granted by Dr. Charlie Degui Chen (Chinese Academy of Sciences). The transfections were performed with Human T cell Nucleofector kit and a Nucleofector (Amaxa), following the protocol provided by the manufacturer. The transfected Tfh cells were cultured in human T cell culture medium. After 24 h of transfection, the culture medium was added 5.0 μg/mL anti-CD28 and 5.0 μg/mL anti-CD3 antibodies for 48 h to stimulate Tfh cells and activate HPK1.

Tfh-B Cell Costimulation
Assays. After isolation, peripheral B cells were cultured in RPMI 1640 medium containing 100 U/mL penicillin G, 50 μg/mL streptomycin, and 10% FBS. 72 h after transfection, Tfh cells and autologous B cells were cocultured in 96-well U-bottom plates at 37°C with 5% CO 2 atmosphere, and the ratio of Tfh cells to B cells was 1 : 10. 50 μL medium was supplemented in every well on day 4, and the supernatants were collected on day 8 to examine IgM, IgG1, IgG2, and IgG3 concentrations. 2.12. Statistical Analysis. Data were expressed as mean ± standard deviation (SD). The difference between SLE patients and healthy controls was compared with twosample t test, and the results of different transfections were compared by paired-sample t test. The data from every control-siRNA group member were set as 1, and the multiple of every JMJD3-siRNA group member relative to its homologous control-siRNA group member was calculated. The results of blank plasmid group and JMJD3-expressing plasmid group were dealt with in the same way. The strength of correlation was analyzed using Pearson's correlation coefficient. P < 0:05 was considered statistically significant. The statistical analysis was performed by the SPSS 25.0 software (SPSS Inc.).

H3K27me3
Expression Increased at the HPK1 Promoter in Tfh Cells of SLE Patients. We combined ChIP with qPCR assays to detect the H3K27me3 expression at the HPK1 promoter region in Tfh cells from SLE and control groups. The results confirmed that the H3K27me3 enrichment at the HPK1 promoter region in SLE Tfh cells was significantly higher than that in the control group (Figure 1(a)).
In order to verify the correlation between H3K27me3 abundance and HPK1 expression, we detected HPK1 mRNA and protein levels in Tfh cells from the SLE group by qPCR and western blot analysis, respectively. The results revealed that H3K27me3 number in the promoter region was significantly negatively correlated with mRNA (Figure 1(b)) and protein (Figure 1(c)) levels of HPK1.

JMJD3 Binding Decreased at the HPK1 Promoter in Tfh
Cells of SLE Patients. In order to explore the cause of the increased H3K27me3 enrichment at the HPK1 promoter in SLE Tfh cells, the levels of EZH2, JMJD3, and UTX at the HPK1 promoter in Tfh cells from the SLE and control groups were measured by combining ChIP and qPCR assays. It was found that JMJD3 binding in the SLE group was greatly lower than that in the control group (Figure 2(a)), and there was no obvious difference in the quantities of EZH2 (Figure 2(b)) and UTX (Figure 2(c)) between the two groups. It was also revealed that the JMJD3 binding was negatively correlated with H3K27me3 enrichment at the HPK1 promoter in Tfh cells of the SLE group (Figure 2(d)), while it was positively correlated with the mRNA (Figure 2(e)) and protein (Figure 2(f)) levels of HPK1.
Studies have confirmed that H3K27 and H3K4 methylations are mutually exclusive [22]. It has also been found that UTX not only demethylates H3K27 but also recruits the MLL family of H3K4 methyltransferases, resulting in increased H3K4 methylation [27][28][29]. However, the relationship between JMJD3 and H3K4 methylation has not been studied. The transfected Tfh cells were assessed in the present study, and it was found that H3K4me3 proportion at the HPK1 promoter in the JMJD3-siRNA group sharply diminished (Figure 3(e)). The MLL1 binding in the region was also markedly reduced (Figure 3(f)), while the levels of MLL2, MLL3, and MLL4 did not statistically change (Figure 3(f)).
We further analyzed the influences of JMJD3 on Tfh cell activity. After 72 h of transfection, the proliferation activities of Tfh cells were assessed by MTT assays, and the quantities of IL-21, BAFF, IFNγ, and IL-17A in the supernatants of Tfh cells were examined by ELISA. Another part of Tfh cells transfected with siRNA was cocultured with autologous B cells for 8 days, whereafter the abundances of IgM, IgG1, IgG2, and IgG3 in the supernatants of cocultures were analyzed using ELISA. Compared to the control group, the proliferation activity of the JMJD3-siRNA group upregulated markedly (Figure 3(g)), the amounts of IL-21 and IFNγ also   7 Journal of Immunology Research increased significantly, but BAFF and IL-17A did not alter profoundly (Figure 3(h)). And after coculturing, IgG1 and IgG3 oversecreted in the JMJD3-siRNA group, while there were no obvious differences in IgM and IgG2 levels between these two groups (Figure 3(i)).

Upregulation of JMJD3 in SLE Tfh Cells Elevated HPK1
Expression, Increased H3K4me3 and MLL1 Amounts in the HPK1 Promoter Region, and Inhibited Tfh Cell Activity. Whereafter, we transfected Tfh cells from three SLE patients with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid. Expectedly, after 72 h of transfection, the protein levels of JMJD3 and HPK1 (Figures 4(a) and  4(b)) and the enrichments of JMJD3 (Figure 4(c)) and H3K4me3 (Figure 4(e)) at the HPK1 promoter all elevated greatly in SLE Tfh cells transfected with the JMJD3overexpressing plasmid. The abundance of H3K27me3 lowered markedly (Figure 4(d)), and MLL1 binding within the HPK1 promoter region increased obviously (Figure 4(f)), while there were no significant differences in abundances of MLL2, MLL3, and MLL4 between the two groups ( Figure 4(f)). Meanwhile, the proliferation activities of Tfh

Discussion
Due to its role in immune regulation, HPK1 has been found to play an important role in various autoimmune diseases, tumors, and inflammatory diseases. Our previous study also confirmed that underexpression of HPK1 led to the excessive activation of Tfh cells in SLE patients, resulting in the intensified inflammatory response [17]. In order to explore the reasons for the reduced HPK1 in SLE Tfh cells, the H3K27me3 enrichment in HPK1 promoter region was detected, and it was found that the H3K27me3 number in this region of Tfh cells from SLE patients increased strikingly, and it was negatively correlated with the mRNA and protein expressions of HPK1. These results suggest that the elevated H3K27me3 level at the promoter leads to the inhibition of HPK1 in Tfh cells of SLE patients.
H3K27me3 amount is regulated by both histone methyltransferase and histone demethylase. The increased H3K27me3 enrichment at the HPK1 promoter in SLE Tfh cells suggested that the levels of these enzymes may be altered. Therefore, we further studied EZH2, JMJD3, and UTX bindings at HPK1 promoter in the two groups. The results showed that JMJD3 number at the HPK1 promoter region in SLE Tfh cells was reduced, and it was negatively correlated with H3K27me3 enrichment, while it positively correlated with HPK1 expression. There were no significant differences in EZH2 and UTX levels between the two groups. These results indicate that the decreased JMJD3 binding in the promoter region contributes to upregulated H3K27me3 level and ultimately inhibits HPK1 expression in SLE Tfh cells.
In order to further confirm the regulatory effects of JMJD3 on HPK1, JMJD3-siRNA was used to downregulate JMJD3 level in Tfh cells from healthy donors. The results revealed that JMJD3 binding at HPK1 promoter alleviated, H3K27me3 enrichment in the region upregulated, and HPK1 expression was profoundly lowered in the JMJD3-siRNA group. Concordantly, the use of JMJD3overexpressed plasmid to increase JMJD3 amount in SLE Tfh cells produced opposite results. The data suggest that JMJD3 regulates HPK1 expression in Tfh cells, and this regulation is achieved at least in part by altering JMJD3 and H3K27me3 levels in the HPK1 promoter region.
Will the change in JMJD3 abundance affect HPK1 quantity by other way and regulate Tfh cell activity at last? We observed that H3K4me3 and MLL1 levels were reduced after downregulating JMJD3 at HPK1 promoter in Tfh cells from healthy controls. Meanwhile, the Tfh cells proliferated greatly, secreted more IL-21 and IFNγ, and stimulated autologous B cells to produce more IgG1 and IgG3. However, overexpression of JMJD3 at HPK1 promoter in SLE Tfh cells had the opposite effects. These results suggest that the attenuate JMJD3 binding at the HPK1 promoter in SLE Tfh cells not only elevates H3K27me3 number but also blocks the recruitment of MLL1, leading to downmodulated H3K4me3 enrichment. All these may further lead to inhibited HPK1 expression and result in Tfh cell overactivation ultimately. As the transfection not only altered JMJD3 binding at the HPK1 promoter but also changed overall JMJD3 expression, we cannot rule out the possibility that JMJD3 can also regulate the levels of MLL1, H3K4me3, HPK1, and Tfh activity through other pathways.
According to the transfection results, we detected H3K4me3 and MLL1 levels at the HPK1 promoter region in Tfh cells from the aforementioned SLE and control groups. The results indicated that MLL1 and H3K4me3 enrichments at HPK1 promoter in SLE Tfh cells were sharply alleviated, and JMJD3 binding at this region was positively correlated with MLL1 and H3K4me3 abundance. These results reconfirm that JMJD3 at the HPK1 promoter in Tfh cells of SLE patients may regulate MLL1 and H3K4me3 numbers, thereby influencing HPK1 expression and Tfh cell activity.

Conclusions
In summary, our results show that the decreased JMJD3 at the HPK1 promoter in Tfh cells from SLE patients leads to upregulation of H3K27me3 in this region and inhibits recruitment of MLL1, thereby downregulating H3K4me3 enrichment. These factors lead to HPK1 underexpression and Tfh cell overactivation and promote the onset and development of SLE in the end. Our findings reveal the epigenetic mechanisms of regulating HPK1 expression in SLE Tfh cells and provide new ideas for the treatment of SLE.

Data Availability
All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author on reasonable request.